REGULATION OF INOSITOL LIPIDS BY THE T CELL MOLECULE CD5

CD 5是一种淋巴细胞特异性的细胞表面分子， 抗原受体所必需的辅助信号 (CD3/Ti）介导的静息外周T细胞活化 淋巴细胞 通过CD 3/Ti的信号转导涉及 膜聚磷酸肌醇（PPI）的水解 肌醇磷酸（InsPs）和二酰基甘油的产生。 我们 观察到CD 5的扰动实质上增加了CD 3/T1， 介导的InsPs的产生，并允许一个短暂的 持续InsP反应。 在没有其他刺激的情况下， CD 5的扰动不改变InsP水平，但确实具有 对磷脂酰肌醇磷酸（PIP）的选择性作用， 二磷酸盐（PIP 2）。 这些观察结果表明，CD 5可能 增加PI激酶或PIP激酶的活性， 从而增加PPI水平并增加CD 3/Ti- 诱导产生InsPs。 为了验证这一假设， 刺激CD 5和CD 3/Ti，单独或组合， 将检查P1、PIP、PIP 2和甘油二酯的质量。 使用从T细胞制备的膜，我们将确定 刺激CD 5是否能增强32 p的掺入 从（32 p）ATP转化为PIP和PIP 2，以及鸟嘌呤是否 核苷酸可以调节这一过程。 CD 5的可能性 在物理上与PI/PIP激酶相关，并且CD 5可以 与PI/PIP激酶的功能性相互作用将使用 免疫沉淀的CD 5和部分纯化的PI和PIP激酶 准备工作 通过用CD 5免疫非淋巴细胞， 基因，我们将确定CD 5是否可以调节PPI水平， 非淋巴样细胞和CD 5是否可以增加InsP的产生 受CD 3/Ti以外的受体影响。 因为细胞质 CD 5的结构域含有三个酪氨酸残基，我们将 确定CD 5的酪氨酸磷酸化是否发生在 T细胞活化。 如果是这样，苯丙氨酸将被取代， 酪氨酸，和这种取代的后果的能力， 将确定CD 5调节PPI的能力。 的总目标 这些研究是为了了解分子机制， CD 5调节PIP和PIP 2，并提供关于 调节T细胞肌醇磷脂代谢 activation.

CD5, a lymphocyte-specific cell-surface molecule, can provide the accessory signals necessary to permit antigen receptor (CD3/Ti)-mediated activation of resting peripheral T lymphocytes. Signal transduction by CD3/Ti involves the hydrolysis of membrane polyphosphoinositides (PPI) with the generation of inositol phosphates (InsPs) and diacylglycerol. We observe that perturbation of CD5 substantially augments CD3/Ti- mediated generations of InsPs and allows an otherwise transient InsP response to be sustained. In the absence of other stimuli, perturbation of CD5 does not change InsP levels but does have selective effects on phosphatidylinositol phosphate (PIP) and bisphosphate (PIP2). These observations suggest that CD5 may increase the activity of either the PI kinase, or the PIP kinase, thereby increasing the levels of PPI and augmenting CD3/Ti- induced generation of InsPs. To test this hypothesis, the effects of stimulating CD5 and CD3/Ti, individually and in combination, on the mass of PI, PIP, PIP2, and diacylglycerol will be examined. Using membranes prepared from T cells, we will determine whether stimulation of CD5 enhances the incorporation of 32p from (32p) ATP into PIP and PIP2 and whether guanine nucleotides can regulate this process. The possibilities that CD5 is physically associated with a PI/PIP kinase and that CD5 can functionally interact with PI/PIP kinases will be examined using immunoprecitated CD5 and partially purified PI and PIP kinase preparations. By transfecting non-lymphoid cells with the CD5 gene, we will determine whether CD5 can regulate PPI levels in non-lymphoid cells and whether CD5 can augment InsP generation by receptors other than CD3/Ti. Because the the cytoplasmic domain of CD5 contains three tyrosine residues, we will determine whether tyrosine phosphorylation of CD5 occurs during T cell activation. If so, phenylalanine will be substituted for tyrosine, and the consequences of this substitution on the ability of CD5 to regulate PPI will be determined. The general aims of these studies are to understand the molecular mechanisms by which CD5 regulates PIP and PIP2 and to provide insights into the regulation of inositol phospholipid metabolism during T cell activation.